Project Summary/Abstract The gut-lung axis is one of many communication axes between the gut microbiome and extra-intestinal systems, organs, and tissues. This axis has been shown to strongly influence lung immune and inflammatory responses, primarily in allergic asthma inflammation and lung infections. The mechanisms for this communication are speculated to be immune cell reprogramming and/or direct translocation of factors, including microbial ligands. This project focuses on the latter mechanism, namely the direct effects of gut microbiome products that make their way into circulation, on lung tissue. The goals of this proposal are to rigorously investigate 1) the gut-lung axis with a focus on metabolites, such as short-chain fatty acids (SCFAs), and commensal-associated molecular patterns (CAMPs), 2) the lung cells that integrate these gut-derived signals, and 3) the effects of these signals of the responsiveness of the lungs to sterile and infectious injury. Based on our preliminary data, we propose that SCFAs and CAMPs are released by the gut microbiota and travel to the lung where they affect the immunometabolic programming of resident immune and non-immune cells, such as alveolar macrophages (AM) and alveolar type 2 (AT2) cells. In Aim One, we will perform metabolomics analysis of stool and lung tissue as well as intervening tissue and vascular beds. This aim will also assess the role of SCFA-producing microbiota in establishing the lung?s baseline metabolite profile and track the migration of specific CAMPs from the gut to the lung. In Aim Two, we will focus on identifying the lung cells that are capable of sensing both CAMPs and metabolites and how they contribute to the lung adopting a primed ?ready? state or an unprimed ?unready? state vis--vis infection and injury. Furthermore, the effect of these CAMPs and metabolites on the metabolic program of the lung in general and the specific lung cells will be examined. In Aim Three, we will explore ways to manipulate the gut-lung axis through antibiotic use, diet, focused metabolite delivery, and through microbiome transfer or restoration. These studies will attempt to establish conditions whereby microbiome-based therapies may help restore healthy lung immune responsiveness. In summary, this project seeks to understand the role of the gut microbiome and dietary metabolites in maintaining healthy lung immunity. Overall, we anticipate that these studies will help fundamentally increase our knowledge of the direct acting effects of the gut microbiome on lung immune responses and in doing so provide valuable insights towards designing future novel therapeutic options that take advantage of the gut-lung immune axis.